This project was part of a larger effort to develop medical countermeasures against the type of radiation injury that might be sustained during a nuclear accident or an act of radiological terrorism. This explains why selenium was used in a mitigating rather than a prophylactic mode and why radiation was administered as a single dose rather than the fractionated doses typically used in radiation therapy.
The pathobiology of late normal-tissue effects of radiation is not well understood. If one accepts the premise that late effects may be at least in part the result of chronic oxidative stress and/or chronic inflammation (12
), selenium has considerable appeal as a potential mitigating agent, because this trace mineral has anti-inflammatory activity and also plays a key role in selenoprotein-mediated antioxidant defenses (13
). Furthermore, selenium has already proven beneficial in the treatment of severe burns and trauma (14
), which makes it an attractive candidate for the mitigation/treatment of the type of combined injuries that might be expected in nuclear accidents or acts of radiological terrorism.
The results of this study show that escalating dietary selenium supplementation from the previously used 100 μg/day to 200 μg/day substantially improves the mitigating effect of selenium on radiation nephropathy as indicated by improved kidney function and a lower incidence of histopathological abnormalities. The mechanism by which selenium mitigates radiation nephropathy is not yet understood. Selenium is an integral part of selenoproteins, several of which are glutathione peroxidases or thioredoxin reductases that play key roles in antioxidant defense mechanisms (16
). However, several lines of evidence suggest that the mitigation of radiation nephropathy we observed in this study involved processes that went beyond the induction of selenoproteins and beyond the reduction of oxidative stress.
For example, a recent review by Sunde (17
) shows that to the extent that rat selenoproteins have been characterized, a dietary selenium content of about 0.1 μg/g is sufficient to maximally induce enzymatic activities as well as mRNA levels. The selenium content of the chow used for our study was 0.33 μg/g. Therefore, antioxidant selenoproteins were most likely maximally induced in our rats even before the introduction of selenium-supplemented drinking water. It is possible, of course, that among the selenoproteins that have not yet been fully characterized, there are antioxidant selenoproteins with higher selenium requirements. However, it is unlikely that their dietary selenium intake requirements would exceed those of known selenoproteins by 2 orders of magnitude.
Subnormal serum selenium levels have been reported in patients who receive TBI as part of the conditioning regimen for hematopoietic stem cell transplants, and the argument has been made that patients who undergo TBI may have higher selenium intake requirements than healthy individuals (18
). There is also an ongoing debate whether low serum selenium levels after radiation therapy are indeed the result of radiation injury or if they are a consequence of prior chemotherapy, pre-existing nutritional deficiencies, or the presence of a tumor (18
). Our study clearly shows that TBI (in the absence of a tumor, pre-existing nutritional deficiencies, or prior chemotherapy) is sufficient to cause a long-lasting reduction of serum selenium levels. However, the reduction is modest and does not offer a plausible explanation for the large doses of selenium required to successfully mitigate radiation nephropathy.
Our experiments were not designed to distinguish between subnormal serum selenium levels as a result of radiation damage to major sites of selenium metabolism (e.g. liver and kidney) and subnormal selenium levels as a result of reduced selenium uptake. As judged by histological criteria, the gastrointestinal tract of the rat recovers quickly after 10 Gy TBI (1
). However, normal histology does not rule out radiation-induced functional impairments that interfere with the absorption of dietary selenium. The fact that irradiated rats tend to drink more and eat less than nonirradiated controls may also have contributed to lower serum selenium levels after TBI.
Both seleno-L-methionine and sodium selenite can induce selenoproteins, but the chemical and physiological characteristics of the two selenium species are quite different [reviewed in ref. (21
)]. Selenite is absorbed by passive diffusion, is quite reactive, can act as an oxidizing agent (thereby potentially exacerbating oxidative stress), and engages in redox cycling and redox signaling. Seleno-L-methionine, on the other hand, is absorbed by active transport, is more bioavailable than selenite (which gives seleno-L-methionine an advantage with regard to the induction of selenoproteins), and has antioxidant properties. Seleno-L-methionine does not engage in redox cycling, but one of its metabolites –methylselenol – does. Thus, if the mitigation of radiation nephropathy in our model had been a matter of reducing oxidative stress either by scavenging radicals or by inducing antioxidant selenoproteins, supplementation with seleno-L-methionine should have been more effective than supplementation with selenite. If, on the other hand, the mitigation of radiation injury involved redox signaling, selenite would be expected to be more effective. It is possible that the complete lack of a mitigating effect of seleno-L-methionine (but not selenite) on radiation-induced glomerular mesangiolysis reflects a qualitative difference between the mitigating mechanisms of the two selenium species. However, other explanations cannot be ruled out at this point.
Selenium doses used for this study were higher than those used typically for the correction of nutritional deficiencies or the prophylaxis against radiation injury (22
). They were in a range known to cause some chronic toxicity in rats. While we did not notice classic signs of selenium toxicity such as hair loss, diarrhea or neurological problems, several animals had to be euthanized because they had stopped feeding and grooming themselves. When this happened after prolonged periods of supplementation, the affected animals usually had very poor kidney function and sometimes also severe anemia. Anemia is common in advanced radiation nephropathy (25
), and hemolytic anemia is one of the known manifestations of chronic selenium toxicity (26
). Both renal failure and anemia can cause lethargy and loss of appetite, which may explain why the animals refused food and water. Refusals of food and water during the early phases of supplementation were more difficult to explain, because these animals appeared to be healthy. However, it is interesting to note in this context that in one trial of high-dose selenium in cattle, one of 17 steers refused selenium-supplemented corn after 11 days of supplementation but then completed the trial with selenium supplied in capsule form for the balance of the 120-day trial (27
). This suggests that individual animals may occasionally refuse high-dose selenium supplements because they object to the taste of the supplement rather than because of a major selenium-induced health problem.
In our study, sudden refusal of food and water was most often encountered in animals that received both TBI and high-dose selenium supplementation. It was only rarely seen in animals that had been exposed to radiation but were maintained on standard drinking water, and it was never observed in nonirradiated animals on selenium-supplemented (200 μg/day) drinking water. Furthermore, in an ongoing study of selenium supplementation in rats that were exposed to localized radiation (10 Gy, single dose, whole-brain), no animals have died or had to be euthanized despite being on selenium-supplemented (200 μg/day) drinking water for 15–18 months. Taken together, this suggests that animals that were exposed to 10 Gy TBI are less tolerant of high doses of selenium than nonirradiated animals, possibly because of radiation-induced injury to major sites of selenium metabolism such as kidney and/or liver. This reduced tolerance would need to be taken into consideration when high-dose selenium is used for the mitigation of radiation injuries in victims of nuclear accidents or acts of radiological terrorism. Shortening the period of supplementation is one possible way to manage the risk of high-dose selenium interventions. Periodic monitoring of kidney and liver functions as well as hematocrits should also help guard against potentially serious side effects of high-dose selenium interventions.
While this study was designed to develop agents for the mitigation of radiation injuries sustained during nuclear accidents or acts of radiological terrorism, it may also have implications for cancer patients who sustain normal tissue injury as a result of radiation therapy.